Introduction

HPLC

(Res1) To obtain a pure protein sample, a protein must be isolated from all other proteins and cellular components. This can prove to be a difficult task as a single protein often makes up only 1% of the total protein concentration of a cell. Therefore 99% of the protein components of a sample must be removed before it can be classified as pure. A task that is equally challenging is keeping the protein in its active form. When we purify proteins we remove them from their natural environments. As a result, it is necessary to simulate the pH, salt concentration and reducing conditions in which they are normally found. In the process of obtaining an active and pure sample we want to minimize the number of steps taken in order to maximize the yield at the end of the separation. Finally, since proteins are made with the intention of only functioning for a short period of time, it is also critical to obtain our sample as quickly as possible. All these components of protein separations can be successfully achieved by a group of separation methods collectively known as chromatography.

There are several properties of proteins that can be taken advantage of to separate proteins. Different types of chromatography take advantage of different properties. Proteins can be separated by:

size

shape

hydrophobicity

affinity to molecules

charge

A Typical Column

In this chapter several different chromatographic methods will be introduced and described. While the methods outlined below all use different characteristics of proteins to separate proteins from one another, they all utilize an insoluble stationary phase and a mobile phase that passes over it. The mobile phase is commonly a liquid solution. It contains the protein we want to isolate. The stationary phase on the other hand is made up of a grouping of beads, usually based on a carbohydrate or acrylamide derivative, that are bound to ionically charged species, hydrophobic characters, or affinity ligands. Much of the success of chromatography is associated with the selection of an appropriate stationary phase.

In column chromatography, when a protein sample is applied to the column, it equilibrates between the stationary phase and the mobile phase. Depending on the type of chromatography, proteins with certain characteristics will bind to the stationary phase while those lacking the sought characteristics will remain in the mobile phase and pass through the column. For example in ion exchange chromatography, a positively charged protein would bind to a negatively charged stationary phase, while the negatively charge protein will be eluted from the column with the mobile phase. The final step involves displacing the protein from the stationary phase, also known as elution, by introducing a particle which will compete with the protein binding site on the stationary phase. Today various commercial column are readily available, specifically Bio-Rad, Sigma-Aldrich,GE Healthcare and Omnifit offers a wide variety of chromatography column.

The image above is a chromatogram that shows the results of a separation based on signals interpreted by a detector.

tm - the time required for the mobile phase to travel the entire length of the column

tr - the time required for a specific protein to elute from the column